EP2021486A2 - Dispositif de fabrication de composés bio-organiques - Google Patents

Dispositif de fabrication de composés bio-organiques

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Publication number
EP2021486A2
EP2021486A2 EP07777278A EP07777278A EP2021486A2 EP 2021486 A2 EP2021486 A2 EP 2021486A2 EP 07777278 A EP07777278 A EP 07777278A EP 07777278 A EP07777278 A EP 07777278A EP 2021486 A2 EP2021486 A2 EP 2021486A2
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EP
European Patent Office
Prior art keywords
bio
phase
organic compound
organic
compound
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German (de)
English (en)
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EP2021486B2 (fr
EP2021486B1 (fr
EP2021486B9 (fr
Inventor
Neil Stephen Renninger
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Amyris Inc
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Amyris Biotechnologies Inc
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M1/00—Apparatus for enzymology or microbiology
    • C12M1/02—Apparatus for enzymology or microbiology with agitation means; with heat exchange means
    • C—CHEMISTRY; METALLURGY
    • C10—PETROLEUM, GAS OR COKE INDUSTRIES; TECHNICAL GASES CONTAINING CARBON MONOXIDE; FUELS; LUBRICANTS; PEAT
    • C10L—FUELS NOT OTHERWISE PROVIDED FOR; NATURAL GAS; SYNTHETIC NATURAL GAS OBTAINED BY PROCESSES NOT COVERED BY SUBCLASSES C10G OR C10K; LIQUIFIED PETROLEUM GAS; USE OF ADDITIVES TO FUELS OR FIRES; FIRE-LIGHTERS
    • C10L1/00—Liquid carbonaceous fuels
    • C10L1/02—Liquid carbonaceous fuels essentially based on components consisting of carbon, hydrogen, and oxygen only
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M1/00—Apparatus for enzymology or microbiology
    • C12M1/04—Apparatus for enzymology or microbiology with gas introduction means
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M21/00—Bioreactors or fermenters specially adapted for specific uses
    • C12M21/12—Bioreactors or fermenters specially adapted for specific uses for producing fuels or solvents
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M27/00—Means for mixing, agitating or circulating fluids in the vessel
    • C12M27/02—Stirrer or mobile mixing elements
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M27/00—Means for mixing, agitating or circulating fluids in the vessel
    • C12M27/18—Flow directing inserts
    • C12M27/20—Baffles; Ribs; Ribbons; Auger vanes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M41/00—Means for regulation, monitoring, measurement or control, e.g. flow regulation
    • C12M41/12—Means for regulation, monitoring, measurement or control, e.g. flow regulation of temperature
    • C12M41/18—Heat exchange systems, e.g. heat jackets or outer envelopes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M41/00—Means for regulation, monitoring, measurement or control, e.g. flow regulation
    • C12M41/12—Means for regulation, monitoring, measurement or control, e.g. flow regulation of temperature
    • C12M41/18—Heat exchange systems, e.g. heat jackets or outer envelopes
    • C12M41/22—Heat exchange systems, e.g. heat jackets or outer envelopes in contact with the bioreactor walls
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/52—Genes encoding for enzymes or proenzymes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P5/00—Preparation of hydrocarbons or halogenated hydrocarbons
    • C12P5/007—Preparation of hydrocarbons or halogenated hydrocarbons containing one or more isoprene units, i.e. terpenes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P5/00—Preparation of hydrocarbons or halogenated hydrocarbons
    • C12P5/02—Preparation of hydrocarbons or halogenated hydrocarbons acyclic
    • C12P5/026—Unsaturated compounds, i.e. alkenes, alkynes or allenes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/02—Preparation of oxygen-containing organic compounds containing a hydroxy group
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/02—Preparation of oxygen-containing organic compounds containing a hydroxy group
    • C12P7/04—Preparation of oxygen-containing organic compounds containing a hydroxy group acyclic
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02E—REDUCTION OF GREENHOUSE GAS [GHG] EMISSIONS, RELATED TO ENERGY GENERATION, TRANSMISSION OR DISTRIBUTION
    • Y02E50/00—Technologies for the production of fuel of non-fossil origin
    • Y02E50/30—Fuel from waste, e.g. synthetic alcohol or diesel

Definitions

  • FIG 5 is a schematic representation of the conversion of IPP and DMAPP to geranyl pyrophosphate ("GPP"), farnesyl pyrophosphate (“FPP”), and geranylgeranyl pyrophosphate (“GGPP”).
  • GPP geranyl pyrophosphate
  • FPP farnesyl pyrophosphate
  • GGPP geranylgeranyl pyrophosphate
  • Figure 6 shows a map of expression plasmid pMBIS-gpps.
  • FIG. 7 shows a map of expression plasmid Pam00408
  • Figure 8 shows a map of expression plasmid pAM424.
  • Figure 9 shows a map of expression plasmids pTrc99A-ADS, pTrc99A-FSA, pTrc99A-LLS, pTrc99A-LMS, pTrc99 A-GTS, pTrc99A-APS, pTrc99A-BPS, pTrc99A-PHS, pTrc99A-TS, pTrc99A-CS, pTrc99 A-SS 5 and pAM373.
  • Figure 10 are schematics for the construction of plasmids pAM489-pAM498.
  • Bio-organic compound refers to an organic compound having at least five carbon atoms that can be made by a host cell by taking a carbohydrate carbon source and converting the carbohydrate carbon source into the desired product.
  • DXP pathway Deoxyxylulose 5-phosphate pathway or "DXP pathway” is used herein to refer to the pathway that converts glyceraldehyde-3 -phosphate and pyruvate to IPP and DMAPP.
  • the DXP pathway is illustrated schematically in Figure 4.
  • Heterologous nucleic acid refers to a nucleic acid wherein at least one of the following is true: (a) the nucleic acid is foreign ("exogenous") to (that is, not naturally found in) a given host cell; (b) the nucleic acid comprises a nucleotide sequence that is naturally found in (that is, is “endogenous to") a given host cell, but the nucleotide sequence is produced in an unnatural (for example, greater than expected or greater than naturally found) amount in the cell; (c) the nucleic acid comprises a nucleotide sequence that differs in sequence from an endogenous nucleotide sequence, but the nucleotide sequence encodes the same protein (having the same or substantially the same amino acid sequence) and is produced in an unnatural (for example, greater than expected or greater than naturally found) amount in the cell; or (d) the nucleic acid comprises two or more nucleotide sequences that are not found in the same relationship to
  • Het cell and "microorganism” are used interchangeably herein to refer to any archae, bacterial, or eukaryotic living cell into which a heterologous nucleic acid can be or has been inserted.
  • the term also relates to the progeny of the original cell, which may not necessarily be completely identical in morphology or in genomic or total DNA complement to the original parent, due to natural, accidental, or deliberate mutation.
  • Isoprenoid and “isoprenoid compound” are used interchangeably herein and refer to a compound derivable from isopentenyl diphosphate.
  • Isolate and "isolating" when referred to a bio-organic compound is the enrichment of the amount of the bio-organic compound in a composition. Consequently, the amount of the bio-organic compound in a composition after the bio-organic compound has been isolated or subject to an isolating step is greater than the amount present in the composition prior to such step.
  • MEV pathway or "MEV pathway” is used herein to refer to the biosynthetic pathway that converts acetyl-CoA to IPP.
  • the MEV pathway is illustrated schematically in Figure 3.
  • a composition that is a "substantially pure" compound is substantially free of one or more other compounds, i.e., the composition contains greater than 80 vol.% , greater than 90 vol.%, greater than 95 vol.%, greater than 96 vol.%, greater than 97 vol.%, greater than 98 vol.%, greater than 99 vol.%, greater than 99.5 vol.%, greater than 99.6 vol.%, greater than 99.7 vol.%, greater than 99.8 vol.%, greater than 99.9 vol.% of the compound; or less than 20 vol.%, less than 10 vol.%, less than 5 vol.%, less than 4 vol.%, less than 3 vol.%, less than 2 vol.%, less than 1 vol.%, less than 0.5 vol.%, less than 0.1 vol.%, or less than 0.01 vol.% of the one ore more other compounds, based on the total volume of the composition.
  • vessel 100 shown in Figure 1 may be closed or under positive pressure during the fermentation process.
  • vessel 100 can be used as an open system whereby one or more of the ports and outlet are opened to the atmosphere providing a system for gas/liquid mass transfer (air or oxygen in and carbon dioxide out).
  • gas outlet 140 may function both as a gas outlet and as a gas inlet where oxygen or air or other gas may be introduced into the system.
  • vessel 100 includes separate gas inlets and separate gas outlets. In such open systems, additional hardware may be included on the vessel for preventing contamination or infiltration of other organisms or other materials into the vessel during the fermentation.
  • FIG. 1 Another vessel embodiment is illustrated in Figure 2. In addition to inlet port
  • agitator 250 may comprise a motor-driven shaft 252 which may include a shaft seal 251 and is connected to one or more impellers 254. Agitator 250 may be typically attached to the top or bottom of the vessel 200.
  • each impeller 254 may be terminated with one or more paddles 256.
  • Impellers 254 may be any suitable shape and may be selected specifically to control amount of mixing, growth rate of the host cells, production rate of the bio-organic compound, shear rate and oxygen or other gas transfer rates.
  • one or more baffles 258 can be added to the vessel 200 to further improve mixing.
  • agitation may be supplied in the form of a recycle line with a pump that draws material from one portion of the vessel such as the bottom and reintroduces the material into the vessel at another portion of the vessel such as the top. Agitation within the vessel of the host cells and the fermentation medium aids in ensuring that the host cells are exposed to adequate nutrients to enable them to grow and produce the bio-organic compounds.
  • oxygen or air can be bubbled through a sparger 260 for improved gas/liquid mass transfer.
  • the sparger 260 may include one or more gas outlets (not shown) that are submerged within the fermentation media, preferably at or near the bottom of the vessel.
  • the sparger 260 may be a sparging ring having multiple gas outlets arranged in a generally circular or round configuration.
  • passive aeration of the vessel may be provided, such as use of various aeration screens, membranes, fibers or other passive aeration devices or by removing a portion of the media from the vessel, oxygenating it and returning it to the vessel.
  • one or more vessels continuously receive host cells, fermentation medium, and optional additives while continuously discharging host cells, byproducts, and/or bio-organic compounds from the vessels.
  • the discharge from one vessel may be used as the feedstock to another vessel that optionally also receives fresh host cells, fermentation medium, nutrients, and/or other additives.
  • a single vessel or a series of vessels together can be configured to provide the desired average residence time for the host cells.
  • a portion of the discharge from one of the down-stream vessels can be returned to one or more upstream vessels to recycle the discharge to an earlier stage of processing, or other materials from processing steps further downstream can be reintroduced into the vessels.
  • the vessels used in some embodiments of the present invention include additional hardware that may be attached to the vessel to facilitate processing.
  • Such hardware may include additional hardware for facilitating clean-in-place and sterilize-in- place processing.
  • one, some or each of the ports, outlets, inlets, valves and all of the hardware inside the vessel may be sterilized in place.
  • the sterilization may occur using steam sterilization.
  • any of the ports, outlets or sampling valves may include or have attached to them additional hardware that provides for steam supply to and condensate return from the port outlet or valve such that it may be steam sterilized prior to use or reuse.
  • the vessel or vessels may have a capacity of at least 100 liters.
  • the vessel or vessels may include or have attached to them sensors and probes for measuring various parameters such as pressure, pH, dissolved oxygen concentration, temperature, gas flow rates, liquid flow rates, liquid level, valve positions, foaming, agitation, power, voltage and any other parameters useful in controlling or optimizing the growth of the host cells and the production of the bio-organic compound or compounds.
  • the sensors and probes may feed information to one or more automated systems for controlling and recording the various parameters measured and for adjusting any of the various parameters by controlling air flowrates, power, heating or cooling to control vessel temperature, stirring rpms, pumps, sterilization or clean in place of the vessel or any of the inlet, outlet, addition, sampling valves or other ports, outlet flow control or any other relevant mechanism for controlling a parameter or parameters of the fermentation.
  • Such adjustments may occur using any known control mechanism, such as for example, control or actuation of various valves, pumps or motors and may use proportional, proportional-integral or proportional —integral- derivative control systems.
  • Any of the vessels used in the production of the bio-organic compounds may include additional hardware, such as additional agitators, additional inlet ports, outlet ports, sampling ports, additional heating/cooling equipment, such as additional heating coils, additional aeration equipment such as additional spargers, additional sensors and probes, additional cleaning or sterilization equipment to facilitate processing or any other parameter of the fermentation.
  • additional hardware such as additional agitators, additional inlet ports, outlet ports, sampling ports, additional heating/cooling equipment, such as additional heating coils, additional aeration equipment such as additional spargers, additional sensors and probes, additional cleaning or sterilization equipment to facilitate processing or any other parameter of the fermentation.
  • an isoprenoid production system which comprises: a. at least one vessel having a capacity of at least 100 liters; b. an aqueous medium, within the at least one vessel, comprising a first phase; c. a plurality of host cells, within the aqueous medium, capable of making, producing or synthesizing one or more isoprenoid compounds; and, d. a liquid organic second phase comprising the one or more isoprenoid compounds in contact with the first phase.
  • the isoprenoid compound or compounds is a C 5 isoprenoid. These compounds are derived from one isoprene unit and are also called hemiterpenes. An illustrative example of a hemiterpene is isoprene. In other embodiments, the isoprenoid compound or compounds is a C 10 isoprenoid. These compounds are derived from two isoprene units and are also called monoterpenes. An illustrative example of a monoterpene is myrcene. In other embodiments, the isoprenoid compound or compounds is a C 15 isoprenoid.
  • the isoprenoid compound or compounds is a C 20 isoprenoid.
  • the isoprenoid compound or compounds is a C20 + isoprenoid.
  • These compounds are derived from more than four isoprene units and include: triterpenes (C30 isoprenoid compounds derived from 6 isoprene units) such as squalene; tetraterpenes (C 40 isoprenoid compounds derived from 8 isoprenoids) such as ⁇ -carotene; and polyterpenes (C 40+ isoprenoid compounds derived from more than 8 isoprene units) such as polyisoprene.
  • the isoprenoid compound or compounds may be any combination of two or more isoprenoid compounds.
  • a method for producing at least one bio-organic compound comprises: a. culturing in an aqueous medium a plurality of host cells that produce, make or synthesize at least one bio-organic compound wherein the aqueous medium comprises a first phase; b. forming a liquid organic second phase comprising the at least one bio- organic compound in contact with the first phase; c. separating at least a portion of the second phase from the first phase; and, d. isolating the at least one bio-organic compound from the second phase.
  • the isoprenoid production system may include one or more additional processing components including: 1) one or more separation systems for separating the at least one bio-organic compound from the aqueous media and the organic second phase; 2) one or more reactors for biologically or chemically altering the at least one bio-organic compound such as by addition, substitution, hydrogenation, alkylation, hydroxylation, condensation, halogenation or any other suitable reaction; 2) one or more blending vessels or systems for blending the at least one bio-organic compound with one or more additional components; 3) and one or more additional purification or separation systems for further purifying the bio-organic composition or the at least one bio-organic compound.
  • additional processing components including: 1) one or more separation systems for separating the at least one bio-organic compound from the aqueous media and the organic second phase; 2) one or more reactors for biologically or chemically altering the at least one bio-organic compound such as by addition, substitution, hydrogenation, alkylation, hydroxylation, condensation, halogenation or any other suitable reaction;
  • the second phase may comprise the at least one bio-organic compound.
  • the bio-organic compound can form a portion, most, or substantially all of the second phase.
  • the bio-organic compound forms 1% to 99%, such as 5% to 95%, 10% to 90%, 20% to 80%, 25% to 75%, 35% to 65%, or 40% to 50% of the second phase.
  • the second phase consists essentially of the bio-organic compound.
  • the plurality of host cells includes more than one type of host cell, such as more than one species or strain of host cells, for example 2-5 species or strains of host cells, for example 2, 3, 4 or 5 species or strains of host cells.
  • the plurality of host cells may produce more than one bio-organic compound, such as 2-5 bio-organic compounds, for example 2, 3, 4, or 5 bio-organic compounds.
  • the bio-organic compound or compounds may be isolated from the first phase and/or second phase using any suitable separation method.
  • the bio- organic compound is isolated from the second phase such that it is substantially pure.
  • the organic second phase occurs spontaneously as a result of chemical and molecular interactions such as differences in solubility, or hydrophobicity, density, concentration or any other spontaneous phase separation mechanism.
  • phase separation of the first and second phases is induced in a separation vessel or vessels or system that may be the same or a different vessel or vessels or processing system as the fermentation vessel or vessels.
  • phase separation is induced by centrifugation such as continuous or batch centrifugation.
  • phase separation is induced by the introduction of a deemulsif ⁇ er or a nucleating agent into the fermentation reaction.
  • a deemulsifier prevents or limits the amount of the bio-organic compound or compounds that emulsify with the aqueous phase.
  • deemulsifiers include flocculants and coagulants.
  • the separate phases can be individually drawn from the separation vessel. Any amount of the second phase can be separated from the first phase, e.g. all, a portion, 1% to 100% such as 5% to 95%, 10% to 90%, 20% to 80%, 25% to 75%, 35% to 65%, or 40% to 50% of the second phase may be separated from the first phase. If the organic second phase is less dense than the aqueous first phase, then one or more taps can be provided or placed on the separation vessel near the interface between the two phases (preferably within the organic second phase) to decant the organic second phase before removing the denser aqueous phase.
  • the aqueous first phase can be removed from the separation vessel using an outlet near the bottom of the separation vessel until the organic second phase appears. At which point, the organic second phase can be transferred into a separate location for further processing or storage. Both of the aqueous first and organic second phases can flow out of the separation vessel under the force of gravity, gas pressure or through the use of a pump or pumps or a combination thereof. [0060] If the organic second phase is denser than the aqueous first phase, then one or more taps can be provided or placed on the separation vessel near the interface between the two phases (preferably within the organic second phase) to decant the aqueous first phase before removing the denser organic second phase.
  • the organic second phase may be removed from the separation vessel using an outlet near the bottom of the separation vessel.
  • a separation vessel with one or more taps can contain a specified volume of the fermentation medium and host cells, and the continually-produced organic second phase may be decanted through the taps to storage or further processing. If the organic second phase is denser than the aqueous first phase, the organic second phase can be removed continuously from the bottom of the separation vessel at a rate that prevents complete depletion of the organic second phase from the separation vessel to avoid drawing from the aqueous first phase.
  • the bio-organic compound may be isolated from the organic second phase using adsorption, a process in which molecules move from a bulk liquid onto the surface of adsorbents.
  • adsorbents include activated carbon; aluminas; aluminosilicates such as zeolites; clays such as fuller's earth; molecular sieves; organic polymers such as polystyrene and resins; and silicas such silica gel.
  • the adsorbent may be used to capture the desired bio- organic product or unwanted byproducts. Isolation by adsorption may be performed using a batch, continuous or semi-continuous process.
  • the bio-organic compound may be isolated from the organic second phase using distillation, a method of separating substances based on differences in their volatilities.
  • batch distillation an entire batch of liquid is initially charged to a vessel and then heated or reduced in pressure within the vessel. Vapor is thereby continuously generated and may be condensed to form a liquid distillate which is collected.
  • continuous equilibrium distillation a continuously flowing liquid feed is heated or reduced in pressure so as to cause partial vaporization of the mixture and separate recovery of liquid and vapor components. The liquid and vapor disengage while flowing through a distillation column, and the products emerge as vapor and liquid streams. When the vapor and liquid approach phase equilibrium, this is called a flashing process. If desired, the vapor product can be condensed to form a liquid distillate.
  • the bio-organic compound or compounds are isolated from the organic second phase using gas-liquid extraction.
  • This process is also known as stripping and is the transfer of a component dissolved in a liquid stream into a vapor stream in a more concentrated form. Temperature and pressure can be optimized for the transfer of the desired bio-organic compound.
  • Illustrative examples of vapor streams include air and steam. Typically, the liquid stream flows down a column while the vapor stream is bubbled up (flowing countercurrently to the liquid stream).
  • the bio-organic compound is isolated from the organic second phase using liquid-liquid extraction. Also known as solvent extraction, liquid-liquid extraction is the transfer of a substance from one liquid phase into another immiscible liquid phase.
  • the feed liquid (the organic second phase) is mixed with a second immiscible liquid phase in a suitable vessel.
  • the mixture is then permitted to settle into layers and separate into extract and raffinate and the lighter layer can be decanted from the vessel.
  • the desired bio-organic compound or compounds can be in the extract or raffinate depending on the product and solvent used.
  • the bio-organic compound is isolated from the organic second and /or the aqueous first phase using ultrafiltration, a pressure-driven membrane process used to separate solution components on the basis of molecular size and shape. Under an applied pressure difference across an ultrafiltration membrane, solvent and small solute species pass through the membrane and are collected as permeate while larger solute species are retained by the membrane and recovered as a concentrated retentate.
  • the host cells are capable of producing from about 50 to about 1500 milligrams, such as more than about 100 milligrams, more than about 150 milligrams, more than about 200 milligrams, more than about 250 milligrams, more than about 500 milligrams, more than about 750 milligrams or more than about 1000 milligrams of bio-organic compound per gram of dry cell weight.
  • the invention comprises a fuel composition production system comprising: a. at least one vessel having a capacity of at least 100 liters; b. an aqueous medium, within the vessel, comprising a first phase; c. a plurality of host cells, within the aqueous medium, capable of making, producing or synthesizing at least one bio-organic compound; and, d. a liquid organic second phase comprising the at least one bio-organic compound in contact with the first phase.
  • the fuel additive is selected from the group consisting of oxygenates, antioxidants, environmental protectants, thermal stability improvers, cetane improvers, stabilizers, cold flow improvers, combustion improvers, anti-foams, anti-haze additives, corrosion inhibitors, lubricity improvers, icing inhibitors, injector cleanliness additives, smoke suppressants, drag reducing additives, metal deactivators, dispersants, detergents, deemulsif ⁇ ers, dyes, markers, static dissipaters, biocides and combinations thereof.
  • the fuel composition production system comprises: a) one or more batch, fed-batch or continuous flow fermentation systems comprising: i) at least one vessel having a capacity of at least 100 liters; ii) an aqueous medium, within the at least one vessel, comprising a first phase; iii) a plurality of host cells, within the aqueous medium, capable of making, producing or synthesizing at least one bio-organic compound; and, iv) a liquid organic second phase comprising the at least one bio- organic compound in contact with the first phase; b) one or more first phase separation systems whereby the first phase and the second organic phase or one or more components of the second organic phase are separated; c) optionally one or more second phase separation systems whereby the at least one bio-organic compound is separated from the second organic phase; d) optionally one or more reactors or vessels wherein the at least one bio- organic compound is chemically or biologically modified; e) optionally one or more purification systems where
  • the one or more first phase separation systems comprises one or more systems, vessels or other phase separation components detailed herein configured specifically to separate the first phase from the second organic phase.
  • the one or more second phase separation systems includes one or more systems, vessels or phase separation components detailed herein configured specifically to separate the bio-organic compound or compounds from the second organic phase.
  • the one or more reactors wherein the at least one bio- organic compound is chemically or biologically modified comprises the same or different vessel or vessels used for the fermentation or the separation systems.
  • the fuel composition system includes one or more blending vessels or systems for blending the at least one bio-organic compound with one or more additional fuel components.
  • the blending vessel or blending system may be any suitable vessel or system.
  • the blending vessel may include any or all of the inlets, outlets, ports, sensors, probes, agitators and other hardware identified for the bio-organic compound production vessel.
  • the blending vessel may blend one or more fuel components with the bio- organic compound or compounds. For example, 2-5 fuel components, such as 3 or 4 fuel components.
  • the blending system may be batch, continuous or fed batch.
  • the invention comprises a method of making a fuel composition comprising: a.
  • the biofuel further comprises at least one bio-organic compound and a petroleum-based fuel, a fuel additive or a combination thereof.
  • the petroleum-based fuel is a gasoline, jet fuel, kerosene, diesel fuel or a combination thereof.
  • the bio-organic compound production system or the fuel composition production system may be built or created by retrofitting an ethanol production facility.
  • any suitable host cell can be used in the practice of the present invention.
  • the host cell is a genetically modified host microorganism in which nucleic acid molecules have been inserted, deleted or modified (i.e., mutated; e.g., by insertion, deletion, substitution, and/or inversion of nucleotides), to either produce the desired bio-organic compound, or effect an increased yield of the desired bio-organic compound.
  • suitable host cells include any archae, bacterial, or eukaryotic cell.
  • archae cells include, but are not limited to those belonging to the genera: Aeropyrum, Archaeglobus, Halobacterium, Methanococcus, Methanobacterium, Pyrococcus, Sulfolobus, and Thermoplasma.
  • Illustrative examples of archae species include but are not limited to: Aeropyrum pernix, Archaeoglobus fulgidus, Methanococcus jannaschii, Methanobacterium thermoautotrophicum, Pyrococcus abyssi, Pyrococcus horikoshii, Thermoplasma acidophilum, Thermoplasma volcanium.
  • eukaryotic species include but are not limited to:
  • Illustrative examples of species with non-pathogenic strains include but are not limited to: Fusarium graminearum, Fusarium venenatum, Pichia pastoris, Saccaromyces boulardi, and Saccaromyces cerevisiae.
  • the host cells of the present invention have been designated by the Food and Drug Administration as GRAS or Generally Regarded As Safe.
  • Illustrative examples of such strains include: Bacillus subtilis, Lactibacillus acidophilus, Lactobacillus helveticus, and Saccharomyces cerevisiae. Engineering Pathways to make Bio-Organic Compounds
  • An illustrative example of a class of bio-organic compounds is isoprenoids.
  • Isoprenoids serve to maintain cellular fluidity, electron transport, and other metabolic functions.
  • isoprenoids are useful as pharmaceuticals, cosmetics, perfumes, pigments and colorants, fungicides, antiseptics, nutraceuticals, and fine chemical intermediates.
  • Isoprenoid compounds are made in nature through two different metabolic pathways which converge at IPP and its isomer, DMAPP.
  • eukaryotes other than plants use the MEV isoprenoid pathway exclusively to convert acetyl-CoA to IPP, which is subsequently isomerized to DMAPP.
  • Prokaryotes use the mevalonate-independent or DXP pathway to produce IPP and DMAPP separately through a branch point.
  • plants use both the MEV and DXP pathways for IPP synthesis.
  • the methods described herein for engineering the MEV and DXP pathways to make the desired isoprenoid compound can be readily adapted to similarly engineer other pathways to make other bio-organic compounds.
  • acetyl-CoA thiolase also known as acetyl-CoA acetyltransferase.
  • nucleotide sequences include but are not limited to the following GenBank accession numbers and the organism from which the sequences derived: (NC_000913 REGION:
  • acetoacetyl-CoA is enzymatically condensed with another molecule of acetyl-CoA to form 3-hydroxy-3-methylglutaryl-CoA
  • HMG-CoA An enzyme known to catalyze this step is, for example, HMG-CoA synthase.
  • KO 3988 (AF542543; Nicotiana attenuata), (AB037907; Kitasatospora griseola), (AX128213, providing the sequence encoding a truncated HMGR; Saccharomyces cerevisiae), and (NC_001145: complement (115734..118898; Saccharomyces cerevisiae).
  • mevalonate is enzymatically phosphorylated to form mevalonate 5-phosphate.
  • An enzyme known to catalyze this step is, for example, mevalonate kinase.
  • Illustrative examples of nucleotide sequences include but are not limited to: (L77688; Arabidopsis thaliana), and (X55875; Saccharomyces cerevisiae).
  • a second phosphate group is enzymatically added to mevalonate 5-phosphate to form mevalonate 5-pyrophosphate.
  • An enzyme known to catalyze this step is, for example, phosphomevalonate kinase.
  • nucleotide sequences include but are not limited to: (AF429385; Hevea brasiliensis), (NM_006556; Homo sapiens), and (NC_001 145. complement 712315..713670; Saccharomyces cerevisiae).
  • nucleotide sequences include but are not limited to: (AF035440; Escherichia coli), (NC_002947, locus tag PP0527; Pseudomonas putida KT2440), (CP000026, locus tag SPA2301 ; Salmonella enterica Paratyphi, see ATCC 9150), (NC_007493, locus tag RSP_0254; Rhodobacter sphaeroides 2.4.1), (NC_005296, locus tag RPA0952; Rhodopseudomonas palustris CGA009), (NC_004556, locus tag PD 1293 ⁇ Xylella fastidiosa TemeculaJ), and (NC_003076, locus tag AT5G11380; Arabidopsis thalian ⁇ ).
  • l-deoxy-D-xylulose-5 -phosphate is converted to 2C- methyl-D-erythritol-4-phosphate.
  • An enzyme known to catalyze this step is, for example, 1- deoxy-D-xylulose-5-phosphate reductoisomerase.
  • 2C-methyl-D-erythritol-4-phosphate is converted to 4- diphosphocytidyl-2C-methyl-D-erythritol.
  • An enzyme known to catalyze this step is, for example, 4-diphosphocytidyl-2C-methyl-D-erythritol synthase.
  • nucleotide sequences include but are not limited to: (AF230736; Escherichia col ⁇ ), (NC_007493, locus_tag RSP_2835; Rhodobacter sphaeroides 2.4.1), (NC_003071, locus_tag AT2G02500; Arabidopsis thaliana), and (NC_002947, locus_tag PP 1614; Pseudomonas putida KT2440).
  • 4-diphosphocytidyl-2C-methyl-D-erythritol-2-phosphate is converted to 2C-methyl-D-erythritol 2, 4-cyclodiphosphate.
  • An enzyme known to catalyze this step is, for example, 2C-methyl-D-erythritol 2, 4-cyclodiphosphate synthase.
  • nucleotide sequences include but are not limited to: (AF230738; Escherichia col ⁇ ), (NC_007493, locus_tag RSP_6071; Rhodobacter sphaeroides 2.4.1), and (NC_002947, locus_tag PP 1618; Pseudomonas putida KT2440).
  • nucleotide sequences include but are not limited to: (AY033515; Escherichia colt), (NC_002947, locus_tag PP0853; Pseudomonas putida KT2440), and (NC_007493, locus_tag RSP_2982; Rhodobacter sphaeroides 2.4.1).
  • l-hydroxy-2-methyl-2-(E)-butenyl-4-diphosphate is converted into either IPP or its isomer, DMAPP.
  • An enzyme known to catalyze this step is, for example, isopentyl/dimethylallyl diphosphate synthase.
  • nucleotide sequences include but are not limited to: (AY062212; Escherichia coli) and
  • NC_002947 locusjag PP0606; Pseudomonas putida KT2440.
  • cross talk between the host cell's own metabolic processes and those processes involved with the production of IPP as provided by the present invention are minimized or eliminated entirely.
  • cross talk is minimized or eliminated entirely when the host microorganism relies exclusively on the DXP pathway for synthesizing IPP, and a MEV pathway is introduced to provide additional IPP.
  • Such host organisms would not be equipped to alter the expression of the
  • MEV pathway enzymes or process the intermediates associated with the MEV pathway enzymes or process the intermediates associated with the MEV pathway.
  • the host cell produces IPP via the MEV pathway, either exclusively or in combination with the DXP pathway.
  • a host's DXP pathway is functionally disabled so that the host cell produces IPP exclusively through a heterologously introduced MEV pathway.
  • the DXP pathway can be functionally disabled by disabling gene expression or inactivating the function of one or more of the naturally occurring DXP pathway enzymes.
  • the host cell produces IPP via the DXP pathway, either exclusively or in combination with the MEV pathway.
  • a host's MEV pathway is functionally disabled so that the host cell produces IPP exclusively through a heterologously introduced DXP pathway.
  • the MEV pathway can be functionally disabled by disabling gene expression or inactivating the function of one or more of the naturally occurring MEV pathway enzymes.
  • Exemplary Cs bio-organic compounds are hemiterpenes which are generally are derived from IPP or DMAPP.
  • An illustrative example of a hemiterpene is isoprene.
  • Isoprene whose structure is is found in many plants. Isoprene is made from IPP by isoprene synthase. Illustrative examples of suitable nucleotide sequences include but are not limited to: (AB198190;
  • Cio bio-organic compounds are monoterpenes which are generally derived from geranyl pyrophosphate (GPP) which in turn is made by the condensation of IPP with DMAPP.
  • GPP geranyl pyrophosphate
  • An enzyme known to catalyze this step is, for example, geranyl pyrophosphate synthase.
  • Figure 5 shows schematically how IPP and DMAPP can produce GPP, which can be further processed to a monoterpene.
  • nucleotide sequences for geranyl pyrophosphate synthase include but are not limited to: (AF513111; Abies grandis), (AF513112; Abies grandis), (AF513113; Abies grandis), (AY534686; Antirrhinum majus), (AY534687;
  • Antirrhinum majus (Y17376; Arabidopsis thaliana), (AE016877, Locus API 1092; Bacillus cereus; ATCC 14579), (AJ243739; Citrus sinensis), (AY534745; Clarkia breweri),
  • Cio compounds include but are not limited to:
  • CitMTSL4 CitMTSL4
  • AY575970 Lotus corniculatus var. japonicus
  • ⁇ -Pinene is made from GPP by ⁇ -pinene synthase.
  • nucleotide sequences include but are not limited to: (+) ⁇ - pinene synthase (AF543530, REGION: 1.-1887; Pinus taeda), (-) ⁇ -pinene synthase
  • ⁇ -Pinene is made from GPP by ⁇ -pinene synthase.
  • suitable nucleotide sequences include but are not limited to: (-) ⁇ -pinene synthases (AF276072, REGION: 1..1749; Artemisia annua) and
  • ⁇ -terpinene is made from GPP by a ⁇ -terpinene synthase.
  • suitable nucleotide sequences include: (AF514286, REGION: 30..1832 from Citrus limon) and (ABl 10640, REGION 1..1803 from Citrus urtshiu).
  • Exemplary C 15 bio-organic compounds are sesquiterpenes which are generally derive from farnesyl pyrophosphate (FPP) which in turn is made by the condensation of two molecules of IPP with one molecule of DMAPP.
  • FPP farnesyl pyrophosphate
  • An enzyme known to catalyze this step is, for example, farnesyl pyrophosphate synthase.
  • Figure 5 also shows schematically how IPP and DMAPP can be combined to produce FPP, which can be further processed to a sesquiterpene.
  • NC_005823 Locus YP_000273; Leptospira interrogans serovar Copenhageni str. Fiocruz Ll-130), (AB003187; Micrococcus luteus), (NC_002946, Locus YP_208768; Neisseria gonorrhoeae FA 1090), (U00090, Locus AAB91752; Rhizobium sp.
  • NGR234 (J05091; Saccharomyces cerevisae), (CP000031, Locus AAV93568; Silicibacter pomeroyi DSS-3), (AE008481, Locus AAK99890; Streptococcus pneumoniae R6), and (NC_004556, Locus NP 779706; Xylella fastidiosa Temeculal).
  • FPP by amorphadiene synthase FPP by amorphadiene synthase.
  • An illustrative example of a suitable nucleotide sequence is
  • Figure 5 shows schematically how IPP and DMAPP can be combined to produce FPP, which can then be further processed to produce amophadiene.
  • ⁇ -Farnesene is made from FPP by ⁇ -farnesene synthase.
  • suitable nucleotide sequences include but are not limited to
  • Nerolidol [00140] Nerolidol, whose structure is
  • Nerolidol is made from FPP by a hydroxylase such as nerolidol synthase.
  • An illustrative example of a suitable nucleotide sequence includes but is not limited to AF529266 from Zea mays (maize; gene tpsl).
  • nucleotide sequence includes but is not limited to AF441124 REGION: L.1647 from Citrus sinensis and AY917195 REGION: 1..1653 from Per ilia frutescerts.
  • Exemplary C2 0 bio-organic compounds are diterpenes which are generally derived from geranylgeraniol pyrophosphate (GGPP) which in turn is made by the condensation of three molecules of IPP with one molecule of DMAPP.
  • An enzyme known to catalyze this step is, for example, geranylgeranyl pyrophosphate synthase.
  • Figure 5 also shows schematically how IPP and DMAPP can be combined to produce GGPP, which can be further processed to a diterpene, or can be further processed to produce a carotenoid.
  • nucleotide sequences for geranylgeranyl pyrophosphate synthase include but are not limited to: (ATHGERPYRS; Arabidopsis thaliana), (BT005328; Arabidopsis thaliana), (NM_119845; Arabidopsis thaliana), (NZ_AAJM01000380, Locus ZP_00743052; Bacillus thuringiensis serovar israelensis, ATCC 35646 sql563), (CRGGPPS; Catharanthus roseus), (NZ_AABF02000074, Locus ZP_00144509; Fusobacterium nucleatum subsp.
  • HGERPYRS Arabidopsis thaliana
  • BT005328 Arabidopsis thaliana
  • NM_119845 Arabidopsis thaliana
  • NZ_AAJM01000380 Locus ZP_00743052
  • lusitanicus (AB016044; Mus musculus), (AABXO 1000298, Locus NCUO 1427; Neurospora crassa), (NCU20940; Neurospora crassa), (NZ_AAKL01000008, Locus ZP_00943566; Ralstonia solanacearum UW551), (ABl 18238; Rattus norvegicus), (SCU31632; Saccharomyces cerevisiae), (ABOl 6095; Synechococcus elongates), (SAGGPS; Sinapis alba), (SSOGDS; Sulfolobus acidocaldarius), (NC_007759, Locus YP_461832; Syntrophus aciditrophicus SB), and (NC_006840 s Locus YP_204095; Vibrio ⁇ scheri ESU4).
  • GGPP can also be made by adding IPP to FPP.
  • Illustrative examples of nucleotide sequences encoding an enzyme capable of this reaction include but are not limited to: (NM_112315; Arabidopsis thalian ⁇ ), (ERWCRTE; Pantoea agglomerans), (D90087, Locus BAA14124; Pantoea ananatis), (X52291, Locus CAA36538;
  • Rhodobacter capsulatus (AF195122, Locus AAF24294; Rhodobacter sphaeroides), and
  • GGPP is then subsequently converted to a variety of C 2 o isoprenoids.
  • Illustrative examples of such compounds include sesterterpenes (C 25 compound made from five isoprene units), triterpenes (C30 compounds made from six isoprene units), and tetraterpenes (C4 0 compound made from eight isoprene units). These compounds are made by using similar methods described herein and substituting or adding nucleotide sequences for the appropriate synthase(s).
  • Engineering Pathways [00151] Although for illustrative purposes, the invention has been described with reference to engineering the MEV and/or DXP pathways, these methods can be adapted to similarly engineer suitable pathways to make non-isoprenoid bio-organic compounds. These pathways are typically engineered using recombinant DNA technology by expression of suitable heterologous sequences encoding one or more enzymes.
  • the enzyme coding region can be carried out by changing the order of the coding regions on the polycistronic mRNA of an operon or breaking up an operon into individual genes, each with its own control elements, or increasing the strength of the promoter (transcription initiation or transcription control sequence) to which the enzyme coding region is operably linked (for example, using a consensus arabinose- or lactose-inducible promoter in an Escherichia coli host microorganism in place of a modified lactose-inducible promoter, such as the one found in pBluescript and the pBBRlMCS plasmids), or using an inducible promoter and inducing the inducible-promoter by adding a chemical to a growth medium.
  • a consensus arabinose- or lactose-inducible promoter in an Escherichia coli host microorganism in place of a modified lactose-inducible promoter, such as the one found in pBluescript and the pB
  • Suitable low-copy (centrorneric) expression vectors for yeast include, but are not limited to, pRS415 and pRS416 (Sikorski & Hieter (1989) Genetics 122:19-27).
  • Suitable high-copy 2 micron expression vectors in yeast include, but are not limited to, pRS425 and pRS426 (Christainson et al. (1992) Gene 110:119-122).
  • Alternative 2 micron, expression vectors include non-selectable variants of the 2 micron vector (Bruschi & Ludwig (1988) Curr. Genet. 15:83-90) or intact 2 micron plasmids bearing an expression cassette (as exemplified in U.S. Pat. Appl.
  • promoters used in expression vectors are inducible. In other embodiments, the promoters used in expression vectors are constitutive. In some embodiments, one or more nucleic acid sequences are operably linked to an inducible promoter, and one or more other nucleic acid sequences are operably linked to a constitutive promoter.
  • apagC promoter (Pulkkinen and Miller, J. Bacteriol. (1991) 173(l):86-93; Alpuche-Aranda et al. (1992) Proc. Natl. Acad. Sci. U S A. 89(21):10079-83), a nirB promoter (Harborne et al. (1992) MoI. Micro. 6:2805-2813), and the like (see, for example, Dunstan et al. (1999) Infect. Immun. 67:5133-5141; McKelvie et al. (2004) Vaccine 22:3243-3255; and Chatfield et al. (1992) Biotechnol.
  • the total activity of a heterologous enzyme that plays a larger role in the overall yield of a bio-organic compound relative to other enzymes in the respective pathways is increased by expressing the enzyme from a strong promoter.
  • Suitable strong promoters for Escherichia coli include, but are not limited to Trc, Tac, T5, T7, and PLam bd a.
  • the total activity of one or more engineered pathway enzymes in a host is increased by expressing the enzyme from a strong promoter on a high copy number plasmid.
  • Escherichia coli include, but are not limited to using Trc, Tac, T5, T7, and P Lambda promoters with pBAD24, pBAD18, pGEM, pBluescript, pUC, and pTZ vectors.
  • a xylose- inducible promoter for example, Pxyl (see, for example, Kim et all (1996) Gene 181:71-76); a GALl promoter; a tryptophan promoter; a lac promoter; an alcohol-inducible promoter, for example, a methanol-inducible promoter, an ethanol-inducible promoter; a raffinose- inducible promoter; a heat-inducible promoter, for example, heat inducible lambda PL promoter; a promoter controlled by a heat-sensitive repressor (for example, CI857-repressed lambda-based expression vectors; see, for example, Hoffmann et al. (1999) FEMS Microbiol Lett. 177(2):327-34); and the like.
  • a heat-sensitive repressor for example, CI857-repressed lambda-based expression vectors
  • Non-limiting examples of suitable constitutive promoters for use in yeast host cells include an ADHl, an ADH2, a PGK, or a LEU2 promoter.
  • suitable inducible promoters for use in yeast host cells include, but are not limited to, a divergent galactose-inducible promoter such as a GAL 1 or a GAL 10 promoter (West at al. (1984) MoI. Cell. Biol. 4(1 1):2467-2478), or a CUPl promoter.
  • the subject vector comprises a promoter that is stronger than a native E. Coli Lac promoter.
  • the expression vector may also contain one or more selectable marker genes that, upon expression, confer one or more phenotypic traits useful for selecting or otherwise identifying host cells that carry the expression vector.
  • selectable markers for eukaryotic cells include dihydrofolate reductase and neomycin resistance.
  • suitable selectable markers for prokaryotic cells include tetracycline, ampicillin, chloramphenicol, carbenicillin, and kanamycin resistance.
  • a suitable plasmid maintenance system for this purpose is encoded by the parDE operon of RK2, which codes for a stable toxin and an unstable antitoxin.
  • the antitoxin can inhibit the lethal action of the toxin by direct protein-protein interaction. Cells that lose the expression vector that harbors the par DE operon are quickly deprived of the unstable antitoxin, resulting in the stable toxin then causing cell death.
  • the RK2 plasmid replication system is encoded by the trfA gene, which codes for a DNA replication protein.
  • the RK2 plasmid segregation system is encoded by the parCBA operon, which codes for proteins that function to resolve plasmid multimers that may arise from DNA replication.
  • nucleic acids can be prepared from the resultant host cells, and the specific sequences of interest can be amplified by PCR using primers specific for the sequences of interest.
  • the amplified product is subjected to agarose gel electrophoresis, polyacrylamide gel electrophoresis or capillary electrophoresis, followed by staining with ethidium bromide, SYBR Green solution or the like, or detection of DNA with a UV detection.
  • nucleic acid probes specific for the sequences of interest can be employed in a hybridization reaction.
  • the yield of a bio-organic compound via one or more metabolic pathways disclosed herein can be augmented by inhibiting reactions that divert intermediates from productive steps towards formation of the bio-organic product. Inhibition of the unproductive reactions can be achieved by reducing the expression and/or activity of enzymes involved in one or more unproductive reactions. Such reactions include side reactions of the TCA cycle that lead to fatty acid biosynthesis, alanine biosynthesis, the aspartate superpathway, gluconeogenesis, heme biosynthesis, and/or glutamate biosynthesis, at a level that affects the overall yield of the bio-organic compound.
  • Expression plasmid pAM36-MevT66 was generated by inserting the MevT66 operon into the pAM36 vector.
  • the MevT66 operon was synthetically generated using the nucleotide sequence SEQ ID NO: 1 as a template, which comprises the atoB gene from Escherichia coli (GenBank accession number NC_000913 REGION: 2324131..2325315), the ERG13 gene from Saccharomyces cerevisiae (GenBank accession number X96617, REGION: 220..1695), and a truncated version of the HMGl gene from Saccharomyces cerevisiae (GenBank accession number M22002, REGION: 1777..3285), all three sequences being codon- optimized for expression in Escherichia coli.
  • the synthetically generated MevT66 operon was flanked by a 5' EcoRI restriction enzyme site and a 3' Hind III restriction enzyme site, and could thus be cloned into compatible restriction enzyme sites of a cloning vector such as a standard pUC or pAC YC origin vector.
  • the MevT66 operon was PCR amplified with flanking Sfil and AsiSI restriction enzyme sites, the amplified DNA fragment was digested to completion using Sfil and AsiSI restriction enzymes, the reaction mixture was resolved by gel electrophoresis, the approximately 4.2 kb DNA fragment was gel extracted using a Qiagen gel purification kit (Valencia, CA), and the isolated DNA fragment was ligated into the Sfil AsiSI restriction enzyme site of the pAM36 vector, yielding expression plasmid pAM36-MevT66.
  • Expression plasmid pAM25 was generated by inserting the MevT66 operon into the pAM29 vector.
  • Vector pAM29 was created by assembling the pi 5 A origin of replication and kan resistance gene from pZS24-MCSl (Lutz and Bujard (1997) Nucl Acids Res. 25:1203-1210) with an oligonucleotide-generated lacUVS promoter.
  • Expression plasmid pMevB-Cm was generated by inserting the MevB operon into the pBBRl MCS-I vector.
  • the MevB operon encodes the set of enzymes that together convert (R)-mevalonate to IPP, namely mevalonate kinase, phosphomevalonate kinase, and mevalonate pyrophosphate carboxylase.
  • the MevB operon was generated by PCR amplifying from Saccharomyces cerevisiae genomic DNA the coding sequences of the ERGl 2 gene (GenBank accession number X55875, REGION: 580..1911) (encodes a mevalonate kinase), the ERG8 gene (GenBank accession number Z49939, REGION: 3363..4718) (encodes a phosphomevalonate kinase), and the MVDl gene (GenBank accession number X97557, REGION: 544..1734) (encodes a mevalonate pyrophosphate carboxylase), and by splicing the PCR fragments together using overlap extensions (SOEing).
  • SOEing overlap extensions
  • the stop codons of ERGl 2 and ERG8 were changed from TAA to TAG during amplification to introduce ribosome binding sites. After the addition of 3' A overhangs, the MevB operon was ligated into the TA cloning vector pCR4 (Invitrogen, Carlsbad, CA).
  • the MevB operon was excised by digesting the cloning construct to completion using Pstl restriction enzyme, resolving the reaction mixture by gel electrophoresis, gel extracting the 4.2 kb DNA fragment, and ligating the isolated DNA fragment into the Pstl restriction enzyme site of vector pBBRl MCS-I (Kovach et al, Gene 166(1): 175-176 (1995)), yielding expression plasmid pMevB-Cm.
  • Expression plasmid pMBI was generated by inserting the MBI operon into the pB BR IMC S -3 vector.
  • the MBI operon encodes the same enzymes as the MevB operon, as well as an isopentenyl pyrophosphatase isomerase that catalyzes the conversion of IPP to DMAPP.
  • Expression plasmid pMBIS was generated by inserting the isp ⁇ gene into pMBI.
  • the ispA gene encodes a farnesyl pyrophosphate synthase that catalyzes the conversion of IPP and DMAPP to FPP.
  • the coding sequence of the ispA gene (GenBank accession number D00694, REGION: 484..1383) was PCR amplified from Escherichia coli genomic DNA using a forward primer with a Sacll restriction enzyme site and a reverse primer with a Sad restriction enzyme site.
  • the amplified PCR product was digested to completion with Sacll and Sad restriction enzymes, the reaction mixture was resolved by gel electrophoresis, and the 0.9 kb DNA fragment was gel extracted.
  • the isolated DNA fragment was ligated into the SacII Sad restriction enzyme site of pMBI, thereby placing the ispA gene 3' of idi and the MevB operon, and yielding expression plasmid pMBIS (see U.S. Patent Number 7,192,751).
  • Expression plasmid pMBIS-gpps was derived from expression plasmid pMBIS by replacing the ispA coding sequence with a nucleotide sequence encoding a geranyl diphosphate synthase ("gpps").
  • a DNA fragment comprising a nucleotide sequence encoding the geranyl diphosphate synthase was generated synthetically using the coding sequence of the gpps gene of Arabidopsis thaliana (GenBank accession number Yl 7376, REGION: 52..1320), codon-optimized for expression in Escherichia coli, as a template.
  • the nucleotide sequence was flanked by a leader SacII restriction enzyme site and a terminal Sad restriction enzyme site, and can be cloned into compatible restriction enzyme sites of a cloning vector such as a standard pUC or pACYC origin vector.
  • the synthetically generated geranyl diphosphate synthase sequence was isolated by digesting the DNA synthesis construct to completion using SacII and Sad restriction enzymes, resolving the reaction mixture by gel electrophoresis, gel extracting the approximately 1.3 kb DNA fragment, and ligating the isolated DNA fragment into the SacII Sad restriction enzyme site of expression plasmid pMBIS, yielding expression plasmid pMBIS-gpps (see Figure 6 for a plasmid map).
  • a DNA fragment comprising a nucleotide sequence encoding the lacUV5 promoter was synthesized from oligonucleotides and sub-cloned into the Ascl Sfil and AsiSI Xhol restriction enzyme sites of pAM43, yielding expression plasmid pAM45. [00191]
  • Example 2 A DNA fragment comprising a nucleotide sequence encoding the lacUV5 promoter was synthesized from oligonucleotides and sub-cloned into the Ascl Sfil and AsiSI Xhol restriction enzyme sites of pAM43, yielding expression plasmid pAM45.
  • the DNA fragment was then partially digested using Spel restriction enzyme, the reaction mixture was resolved by gel electrophoresis, and the 4.8 kb DNA fragment was gel extracted.
  • the isolated DNA fragment was ligated with the S£>e/-digested mvaA PCR product, yielding expression plasmid pAM41.
  • the nucleotide sequence of the atoB(opt):ERGl 3(opt): ⁇ nvaA operon contained in pAM41 is SEQ ID NO: 41.
  • ERGl 3 is also known as HMGS or HMG-CoA synthase.
  • Expression plasmid pAM97 was derived from expression plasmid pAM45 by replacing the MevT66 operon with the (atoB(ppt):mvaS:mvaA) operon of expression plasmid pAM52.
  • Expression plasmid pAM45 was digested to completion using AsiSl and Sfil restriction enzymes, the reaction mixture was resolved by gel electrophoresis, and the 8.3 kb DNA fragment lacking the MevT66 operon was gel extracted.
  • Expression plasmid pAM97 was digested to completion using Sad and Xhol restriction enzymes, the reaction mixture was resolved by gel electrophoresis, the 7.6 kb fragment was gel extracted, and the isolated DNA fragment was ligated with the MBI operon PCR product, yielding expression plasmid pAM97-MBI.
  • Expression plasmid pAM97-MevB was derived from expression plasmid pAM97 and pAM45 by replacing the MBIS operon of pAM97 with the MevB operon of pAM45.
  • the MevB operon was PCR amplified from pAM45 using primers 9-70C (SEQ ID NO: 8) and 26-39A (SEQ ID NO: 10), the reaction mixture was resolved by gel electrophoresis, the 3.9 kb DNA fragment was gel extracted, and the isolated DNA fragment was digested to completion using Sad and Xhol restriction enzymes.
  • Expression plasmid pAM97 was digested to completion using Sad and Xhol restriction enzymes, the reaction mixture was resolved by gel electrophoresis, the 7.6 kb fragment was gel extracted, and the isolated DNA fragment was ligated with the MevB operon PCR product, yielding expression plasmid pAM97-MevB.
  • the RK2 plasmid was digested to completion using Pstl restriction enzyme, the reaction mixture was resolved by gel electrophoresis, the approximately 6.3 kb DNA fragment containing the entire par locus was gel extracted, and the isolated DNA fragment was subcloned into the Pstl restriction enzyme site of the mini RK2 replicon pRRlO (Roberts et al. (1990) JBacteriol.
  • Expression plasmid pAM97 was digested to completion using Asd and S ⁇ cl restriction enzymes, the reaction mixture was resolved by gel electrophoresis, the approximately 9.4 kb DNA fragment was gel extracted, and the isolated DNA fragment was ligated into the MIuI Sad restriction enzyme site of pAM132, yielding expression plasmid pAM128. [00199]
  • Example 3 Example 3
  • the coding sequence of the mvaS gene was PCR amplified from Enterococcus faecalis genomic DNA (ATCC 700802) using 5' phosphorylated primers 4-40 mvaSF BgIII (SEQ ID NO: 13) and 4- 39 mvaSR BamHI (SEQ ID NO: 14), and the PCR product was ligated into the Smal restriction enzyme site of pBlueScripII-KS(+) (Stratagene, La Jolla, CA), yielding expression plasmid pAM 18.
  • Expression plasmid pAM22 was generated by inserting the coding sequence of the mvaE gene of expression plasmid pAM16 into the pZE21-Pnacoi vector.
  • Vector pZE21- P L -iacOi is a derivative of vector pZE21 -MCS-I in which the tet promoter was replaced with the P L -i ac ⁇ i promoter (Lutz and Bujard (1997) Nucl Acids Res. 25:1203-1210).
  • Expression plasmid pAM33 was generated by inserting the coding sequence of the mv ⁇ S gene of expression plasmid pAM18 into expression plasmid pAM22.
  • Expression plasmid pAM18 was digested to completion using BgIII and BamHI restriction enzymes, the reaction mixture was resolved by gel electrophoresis, the approximately 1.2 kb DNA fragment containing the coding sequence of the mvaS gene was gel extracted, and the isolated DNA fragment was inserted into the BamHI site of expression plasmid pAM22, yielding expression plasmid pAM33.
  • Expression plasmid pAM408 was generated by inserting genes encoding enzymes of the "top" DXP pathway into the pAM29 vector.
  • the PCR products were resolved by gel electrophoresis, gel extracted using a Qiagen (Valencia, CA) gel purification kit, digested to completion using appropriate restriction enzymes (Xholan ⁇ Kpnl for the PCR product comprising the dxs gene; Kpnl and Apal for the PCR product comprising the dxr gene; Apal and Ndel for the PCR product comprising the ispD gene; Ndel and MIuI for the PCR product comprising the ispE gene,), and purified using a Qiagen (Valencia, CA) PCR purification kit. Roughly equimolar amounts of each PCR product were then added to a ligation reaction to assemble the individual genes into an operon.
  • Qiagen Valencia, CA
  • the DNA fragments for FSA was amplified by PCR from its DNA synthesis construct using the primer sequences SEQ ID NO: 39 and SEQ ID NO: 40.
  • the resulting PCR product was digested to completion using Ncol and Sad restriction enzymes, the reaction mixture was resolved by gel electrophoresis, the approximately 1.7 kb DNA fragment comprising the ⁇ -farnesene synthase coding sequence was gel extracted, and the isolated DNA fragment was ligated into the NcoISacI restriction enzyme site of the pTrc99A vector, yielding expression plasmid pTrc99A-FSA (see Figure 9 for a plasmid map).
  • the cells were adapted to minimal media by passaging them through 4 to 5 successive rounds of M9-MOPS media containing 0.8% glucose and antibiotics (see Table 2 for the composition of the M9-MOPS medium).
  • the cells were stored at -80 0 C in cryo-vials in 1 mL stock aliquots made up of 400 uL sterile 50% glycerol and 600 uL liquid culture.
  • Seed cultures of host strains B61 and B62 were established by adding a stock aliquot of each strain to 125 mL flasks containing 20 mL M9-MOPS medium, 0.8% % glucose, and antibiotics as detailed in Table 5, and by growing the cultures to saturation.
  • the seed cultures were diluted 1:100 into 140 mL of fresh medium in a 500 mL flask, and grown again to an OD 550 of approximately 0.1, at which point production of amorpha-4,11-diene was induced by adding 140 uL 1 M IPTG to each culture.
  • samples were removed from each culture, and cells were pelleted by centrifugation.
  • HMG-CoA DL-3-hydroxy-3-methylglutaryl coenzyme A sodium salt
  • the assay was started by adding cell lysate, and the disappearance of NADPH was monitored by absorbance at 34OnM. To account for non-specific disappearance of NADPH, results obtained in a control assay lacking HMG-CoA were subtracted from results obtained in test samples. Enzyme activity of the Enterococcus faecalis HMGR was measured similarly except that the assay buffer contained 100 mM potassium phosphate buffer (pH6.5), 0.4 mM NADPH, 1.0 mM EDTA, and 100 mM KCl.
  • a seed culture of host strain B32 was established by adding 0.5 mL of a stock aliquot of the strain to a 250 mL flask containing 50 mL M9-MOPS medium and antibiotics as detailed in Table 1, and by growing the culture overnight at 37 0 C on a rotary shaker at 250 rpm.
  • a seed culture of host strain B32 for fermentation run 060403-3 was established by adding a stock aliquot of the strain to a 250 mL flask containing 50 mL M9- MOPS medium and antibiotics as detailed in Table 1, and by incubating the culture overnight at 37 0 C on a rotary shaker at 250 rpm.
  • the seed culture was used to inoculate at an initial OD ⁇ oo of approximately 1 a 250 mL flask containing 40 mL M9-MOPS medium and antibiotics, and the culture was again incubated at 37°C on a rotary shaker at 250 rpm until it reached an OD 6O O of 3 to 5.
  • Runs 050608-1 and 050629-1 were carried out at 37°C. Airflow in the bioreactor was set at 1-2 L/min; pH was maintained at 7 using ammonium hydroxide and/or sodium hydroxide; initial agitation was 500-600 rprn; foam was controlled with antifoam B (Sigma-Aldich, St. Louis, MO); the dissolved oxygen levels were maintained above 30% using an agitation cascade. After 5-6 hours of cultivation, production of amorpha-4,11-diene by the host cells was induced by adding 0.8 mL of 1 M IPTG to run 050608-1 and 1.2 mL IPTG to run 050629-1. Upon induction, the culture temperature was reduced to 30 0 C. [00253] Run 060403-3 was carried out at 3O 0 C. Airflow in the bioreactor was set at 1-
  • amorpha-4,11-diene concentration in the sample was between 0.63 mg/L and 20 mg/L.
  • amorpha-4,11-diene was captured in the bioreactor by adding 200 mL of an organic overlay to the fermentor at the time of induction.
  • a seed culture of host strain B 153 was established by adding a stock aliquot of the strain to a 250 mL flask containing 50 mL M9-MOPS medium and antibiotics as detailed in Table 1, and growing the culture at 37°C on a rotary shaker at 250 rpm to an OD ⁇ oo of 3.5 to 4.5.
  • amorpha-4,1 1-diene Production of amorpha-4,1 1-diene in the host cells was induced by adding 1 mL of 1 M IPTG to the culture medium. Amorpha-4,1 1-diene was captured and extracted according to two different protocols. In one method, volatile amorpha-4,1 1-diene present in the off-gas was captured by venting the off-gas through a gas-washer containing 200 mL heptanol.
  • amorpha-4,11-diene concentration in the sample was between 0.63 and 20 mg/L.
  • amorpha-4,1 1-diene was captured by adding 200 mL of an organic overlay to the fermentor at the time of induction.
  • Amorpha-4,11-diene was extracted from the culture medium by combining 25 uL broth with 975 uL acetonitrile, shaking the sample at maximum speed on a Fisher Vortex Genie 2TM mixer (Scientific Industries, Inc., Bohemia, NY) for at least 3 minutes, removing cells from the sample by centrifugation, and diluting the acetonitrile solution into ethyl acetate until the amorpha-4.11-diene concentration in the sample was between 0.63 and 20 mg/L.
  • the ethyl acetate samples were analyzed by GC/MS as described in Example 10.
  • Escherichia coli host strain grown under restricted carbon and nitrogen source conditions and at suboptimal temperature.
  • a seed culture of host strain B86 was established by adding a stock aliquot of the strain to a 250 mL flask containing 50 mL M9-MOPS medium and antibiotics as detailed in Table 1. The culture was grown overnight at 37°C on a rotary shaker at 250 rpm, sub- cultured the following morning into the same medium at an O D 6 Oo of approximately 1, and grown again at 37°C and 250 rpm to an OD ⁇ oo of 3 to 5.
  • amorpha-4,11-diene Production of amorpha-4,11-diene in the host cells was induced at an OD 6 Oo of approximately 30 by adding 1 mL of 1 M IPTG to the culture medium.
  • Amorpha-4,11-diene was captured by overlaying the medium with 10% (v/v) of an organic overlay.
  • Amorpha-4,11-diene was then extracted by combining 25 uL of broth with 975 uL methanol, shaking the sample at maximum speed on a Fisher Vortex Genie 2TM mixer (Scientific Industries, Inc., Bohemia, N.
  • This example describes the production of amorpha-4,11 -diene via the DXP pathway in an Escherichia coli host strain.
  • Seed cultures of host strains B003, B617, B618, and B619 were established by adding a stock aliquot of each strain to separate 125 mL flasks containing 25 mL M9-MOPS and antibiotics as detailed in Table 1 , and by growing the cultures overnight. [00273] The seed cultures were used to inoculate at an initial OD ⁇ oo of approximately
  • This example describes the production of 3-methyl-but-3-en-l-ol and 3- methyl-but-2-en-l-ol in Escherichia coli host strains.
  • Seed cultures of host strains B286, B287, B288, and B291 were established by streaking out a stock aliquot of each strain on LB agar containing antibiotics as detailed in Table 1. Three independent colonies were picked for each strain, and each colony was inoculated into 7 mL of LB media containing antibiotics. The cultures were grown overnight at 37°C on a rotary shaker at 250 rpm until late exponential phase.
  • the cultures were then inoculated at an OD ⁇ oo of approximately 0.05, into a 250 mL flask containing 40 ml of M9- MOPS, 2% glucose, 0.5% yeast extract, and antibiotics.
  • the cultures were grown overnight at 37°C on a rotary shaker at 250 rpm until they reached an ODeoo of approximately 0.2, at which point they were induced by adding 40 uL of 1 M IPTG.
  • the cultures were grown for 72 hours at 30°C on a rotary shaker at 250 rpm. One to two times per day, the OD ⁇ oo of each culture was measured, and a 700 uL sample was removed.
  • the temperature program for the analysis was as follows: 60 0 C for 3 minutes, increasing temperature at 60°C/minute to a temperature of 300 0 C, and a hold at 300 0 C for 2 minutes. The total run time was 9 minutes.
  • the resolved samples were analyzed by a Hewlett-Packard model 5973 mass selective detector. Previous mass spectra demonstrated that 3-methyl-3- buten-1-ol and 3-methyl-2-buten-l-ol have a retention time of 2.067 minutes using this GC protocol.
  • Host strain EPY224 was cured of expression plasmid pRS425ADS by growth in YPD medium (Methods in Yeast Genetics: A Cold Spring Harbor Laboratory Course Manual, 2005 ed., ISBN 0-87969-728-8), plating for single colonies on YPD agar, and then patching single colonies onto CSM-Met His agar and CSM-Met Leu agar. Clones that grew on CSM-Met His agar but not on CSM- Met Leu agar were cured (i.e., had lost the plasmid pRS425ADS). One such clone was designated EPY300.
  • EPY300 was transformed with expression plasmid pRS425-ADS- LEU2d, a plasmid identical to pRS425-ADS except that instead of LEU2 it contains a LEU2d selection marker (Erhart and Hollenberg (1983) J. Bacterid. 156: 625-635) yielding host strain Yl 85.
  • Yl 85 host cell transformants were selected on synthetic defined media, containing 2% glucose and all amino acids except histidine, leucine, and methionine (CSM- glucose; MP Biomedicals, Solon, OH).
  • the host strain EPY300 is auxotrophic for leucine biosynthesis (Ieu2), but expression plasmid pRS425-ADS-LEU2d in Yl 85 restores leucine prototrophy (LEU2).
  • Single colonies were patched onto selective medium (CSM-glucose- histidine, leucine, methionine), and grown for 2 days. The cells were scraped from the plate and transferred to 1 mL of 25% (v/v) glycerol in a cryotube. The suspension was mixed, and then stored at -8O 0 C.
  • Seed flasks of host strain Yl 85 were established by adding a stock aliquot of the strain to a 125 mL flask containing 25 mL of CSM-glucose lacking leucine and methionine, and by growing the cultures overnight. The cultures were used to inoculate at an initial ODeoo of approximately 0.05 a 250 mL baffled flask containing 40 mL of synthetic defined media lacking leucine, and containing 0.2% glucose, 1.8% galactose, and 1 mM methionine. The culture was incubated at 30°C on a rotary shaker at 200 rpm.
  • Saccharomyces cerevisiae host strain where the host strain includes a native mevalonate pathway as well as a heterologous mevalonate pathway that is under control of a heterologous regulatory control.
  • Yeast strains CEN.PK2-1C (Y002) (MATA; ura3-52; trpl-289; Ieu2-3,i 12; his3 ⁇ l; MAL2-8C; SUC2) and CEN.PK2-1D (Y003) (MATalpha; ura3-52; trpl-289; Ieu2- 3,112; his3 ⁇ l; MAL2-8C; SUC2) (J. P. van Dijken et al, Enzyme Microb Technol 26, 706 (J un 1 , 2000) were cultivated in either standard rich medium (YPD) or in defined synthetic medium (. D. Rose, F. Winston, P. Heiter, Methods in yeast genetics: a laboratory course manual. (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N. Y., 1990) lacking appropriate nutrients allowing for selection of integrative transformants, plasmid retention, and meiotic progeny.
  • S. cerevisiae strains Y002 and Y003 were prepared for introduction of inducible mevalonate pathway genes by the following.
  • the ERG9 promoter was replaced with the 5. cerevisiae MET3 promoter by PCR amplification of the KanMX-PMET3 region from pAM328 (SEQ ID NO: 43) using primers 50-56-pwlOO-G (SEQ ID NO: 44) and 50-56- pwlOl-G (SEQ ID NO: 45) containing 45 basepairs of homology to the native ERG9 promoter.
  • 10 ⁇ g of the resulting PCR product was transformed into exponentially growing Y002 and Y003 strains using 40% w/w polyethelene glycol 3350 (Sigma-Aldrich St Louis, MO), 100 mM lithium acetate (Sigma), lO ⁇ g Salmon Sperm DNA (Invitrogen) and incubation at 30 0 C for 30 minutes followed by a 42°C heat shock for 30 minutes (as described by Schiestl & Gietz, Curr. Genet. 16: 339 (1989)). Positive recombinants were identified by their ability to grow on rich medium containing 0.5 ⁇ g/ml Geneticin (Invitrogen Co, Carlsbad, CA) and confirmed by diagnostic PCR.
  • the resultant clones were given the designation Y93 (MAT A) and Y94 (MAT alpha).
  • the ADEl open reading frame was replaced with the Candida glabrata LEU2 gene (CgLEU2).
  • the 3.5KB CgLEU2 genomic locus was amplified from C. glabrata genomic DNA (ATCC, Manassas, VA) using primers 61-67-CPK066-G (SEQ ID NO: 46) and 61-67-CPK067-G (SEQ ID NO: 47) containing 50 basepairs of flanking homology to the ADEl open reading frame (ORF).
  • SEQ ID NO: 48 and pAM495 (SEQ ID NO:49), respectively, were digested overnight with Pmel (New England Biolabs, Beverly, MA) and introduced into exponentially growing Y 176 as described above. Positive recombinants were selected for by growth on medium lacking uracil and histidine. Integration into the correct genomic locus was confirmed by diagnostic PCR.
  • plasmid DNA from pAM493 (SEQ ID NO: 52) was digested overnight with Pmel and introduced into exponentially growing diploid cells as described above. Positive recombinants were selected for by growth on medium lacking adenine. Integration into the correct genomic locus was confirmed by diagnostic PCR. The resultant strain was given the designation Y238.
  • Y238 was sporulated in 2% potassium acetate and 0.02% raff ⁇ nose liquid medium.
  • plasmid DNA from ⁇ AM426 (SEQ ID NO:53), containing S 1 . cerevisiae condon optimized Amorphadeine Synthase (ADS) expressed from the S. cerevisiae GALl promoter, was introduced into exponentially growing Y210 and Y211 as described above.
  • S. cerevisiae strains that contained the pAM426 plasmid were selected for by their ability to grow in the absence of leucine supplementation. The resultant strains were given the designation Y225 (MAT A) and Y227 (MAT alpha).
  • ADS Amorphadeine Synthase
  • AMO cytochrome P450 monooxygenase
  • CPR cytochrome P450 oxidoreductase
  • Escherichia coli host strains Escherichia coli host strains.
  • yeast extract 0.5% yeast extract, and antibiotics as detailed in Table 1, and by growing the cultures overnight.
  • Samples were taken every 24 hours up to 120 hours (total of 5 time points) by transferring 2 uL to 10 uL of the organic overlay layer to a clean glass vial containing 1 mL ethyl acetate spiked with trans-caryophyllene as an internal standard.
  • 1 mL aliquots of the cultures were spun down, cell pellets were resuspended in 250 uL sterile water, and the cell suspensions were transferred to a glass vial containing 1 mL ethyl acetate spiked with trans-caryophyllene as an internal standard.
  • the temperature program for the analysis was as follows: 150 0 C hold for 3 minutes, increasing temperature at 25°C/minute to a temperature of 200 0 C, increasing temperature at 60°C/minute to a temperature of 300 0 C, and a hold at 300 0 C for 1 minute.
  • Previous mass spectra demonstrated that the ⁇ -famesene synthase product was ⁇ -farnesene, and that ⁇ -farnesene had a retention time of 4.33 minutes using this GC protocol.
  • Farnesene titers were calculated by comparing generated peak areas against a quantitative calibration curve of purified ⁇ - farnesene (Sigma- Aldrich Chemical Company, St. Louis, MO) in trans-caryophyllene-spiked ethyl acetate.
  • Host strain B592 produced approximately 400 mg/L of ⁇ -farnesene at 120 hours (averaged over 3 independent clones), and had a maximal specific productivity of approximately 46 mg/L/OD ⁇ oo-
  • Host strain B552 produced approximately 1.1 g/L of ⁇ - farnesene at 120 hours (averaged over 3 independent clones), and had a maximal specific productivity of approximately 96 mg/L/OD ⁇ oo (1 representative clone).
  • Example 20 This example describes the production of ⁇ -farnesene via the DXP pathway in an Escherichia coli host strain.
  • Seed cultures of host strains B650, B651, B652, and B653 were established by adding a stock aliquot of each strain to separate 125 mL flasks containing 25 niL M9-MOPS and antibiotics as detailed in Table 1, and by growing the cultures overnight. [00306] The seed cultures were used to inoculate at an initial OD ⁇ oo of approximately
  • Example 21 Samples were taken at various time points by transferring 100 uL samples of the upper organic overlay layer to a clean tube. The tube was centrifuged to separate out any remaining cells or media, and 10 uL of the organic overlay samples were transferred into 500 uL ethyl acetate spiked with beta- or trans-caryophyllene as an internal standard in clean glass GC vials. The mixtures were vortexed for 30 seconds, and then analyzed as described in Example 18. Escherichia coli host strain B653 produced approximately 7 mg/g DCW ⁇ -farnesene. [00307] Example 21
  • This example describes the production of ⁇ -farnesene or ⁇ -farnesene in a
  • Saccharomyces cerevisiae strain EPY224 (Ro et al. (2006) Nature 440: 940-943; PCT Patent Publication WO2007/005604) by culturing in rich medium.
  • Strain EPY300 was then transformed with expression plasmids pRS425-FSA or pR425-FSB, yielding host strains Yl 66.and Yl 64, respectively.
  • the host strain EPY300 was auxotrophic for leucine biosynthesis (leul), but expression plasmid pRS425-FSA or pRS425- FSB restores leucine prototrophy (LEU2).
  • Single colonies were transferred to culture vials containing 5 mL of liquid SM-glu lacking leucine. The cultures were incubated by shaking at 30 0 C until growth reaches stationary phase. The cells were stored at -8O 0 C in cryo-vials in 1 mL frozen aliquots made up of 400 ⁇ L 50% glycerol and 600 ⁇ L liquid culture.
  • Seed cultures were established by adding a stock aliquot to a 125 mL flask containing 25 mL SM-glu lacking leucine, and growing the cultures overnight. The seed cultures were used to inoculate at an initial OD ⁇ oo of approximately 0.05 250 mL baffled flasks containing 40 mL of synthetic defined media lacking leucine, 0.2% glucose, and 1.8% galactose. Cultures were incubated at 30 0 C on a rotary shaker at 200 rpm.
  • Host strain Y166 produced approximately 9.8 mg/L of ⁇ -farnesene at 120 hours (averaged over 3 independent clones), and had a maximal specific productivity of approximately 3 mg/L/OD6oo(l representative clone).
  • Host strain Y164 produced approximately 56 mg/L of ⁇ -farnesene at 120 hours (averaged over 3 independent clones), and had a maximal specific productivity of approximately 20 mg/L/OD ⁇ ooO representative clone).
  • This example describes the production of ⁇ -terpinene, ⁇ -pinene, and terpinolene in Escherichia coli host strains.
  • E. coli DHl-TIr [pMevT, pMevB-Gpps, pAM445]
  • ⁇ -pinene E. coli DHl-TIr [pMevT, pMevB-Gpps, pAM443 or pAM442]
  • terpinolene E. coli DHl-TIr [pMevT, pMevB-Gpps, pAM444] were established by adding a stock aliquot of each strain to separate 125 mL flasks containing 25 mL M9-MOPS, 2% glucose, 0.5% yeast extract, and antibiotics as detailed in Table 1, and by growing the cultures overnight to late exponential phase.
  • the hexadecane overlay was diluted 1:1 or 1:10 with ethyl acetate spiked with trans-caryophyllene as an internal standard in a 1.8 mL GC vial.
  • 1 mL aliquots of the cultures were spun down, cell pellets were resuspended in 250 uL sterile water, and the cell suspensions were transferred to a glass vial containing 1 mL ethyl acetate spiked with trans-caryophyllene as an internal standard.
  • the cell pellets were extracted in the ethyl acetate by vortexing the glass vials for 15 minutes, after which 500 uL of the ethyl acetate extraction was transferred to a clean glass vial.
  • the hexadecane/ethyl acetate samples and the ethyl acetate-extracted cell pellet samples were analyzed on an Agilent 6890N gas chromatograph equipped with an Agilent 5975 mass spectrometer (GC/MS) in full scan mode (50-500 m/z). To expedite run times, the temperature program and column matrix was modified to achieve optimal peak resolution and the shortest overall runtime.
  • Example 23 A 1 ⁇ L sample was split (a split ratio between 1 :2 and 1 :50 was selected based on sample concentration) and then separated using a HP-5MS column (Agilent Technologies, Inc., Palo Alto, CA) and helium carrier gas.
  • the temperature program for the analysis was as follows: 75°C hold for 3 minutes, increasing temperature at 20°C/minute to a temperature of 115°C, increasing temperature at 60°C/minute to a temperature of 30O 0 C, and a hold at 300 0 C for 0.5 minute.
  • the various products, ⁇ -terpinene, ⁇ -pinene, and terpinolene were observed at 5.4, 4.1, 5.4, and 5.9 minutes, respectively. Titers were calculated by comparing generated peak areas against a quantitative calibration curve of purified standards in trans-caryophyllene-spiked ethyl acetate. [00318]
  • Example 23 A 1 ⁇ L sample was split (a split ratio between 1 :2 and 1 :
  • This example describes the production of linalool, limonene, ⁇ -pinene, ⁇ - phellandrene, carene, or sabinine in Escherichia coli host strains.
  • Seed cultures are established by adding a stock aliquot of each strain to separate 125 mL flasks containing 25 mL M9-MOPS, 0.5% yeast extract, 2% glucose, and antibiotics as detailed in Table 1, and by growing the cultures overnight.
  • the seed cultures are used to inoculate at an initial OD ⁇ oo of approximately
  • Primer 67-1 A-C for PCR amplification of the coding sequence of the dxs gene

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Abstract

L'invention concerne un système et un procédé de fabrication de composés bio-organiques. Le système selon l'invention peut comporter une cuve, et une première phase contenant un milieu aqueux renfermant des cellules hôtes capables de produire un composé bio-organique, le composé bio-organique comportant une deuxième phase se trouvant en contact avec le milieu aqueux.
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BRPI0712508A2 (pt) 2012-08-28
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WO2007139924A9 (fr) 2008-03-27
JP2013143944A (ja) 2013-07-25
HN2008001755A (es) 2010-12-27
KR101420889B1 (ko) 2014-07-17
ES2527876T5 (es) 2022-04-26
AU2007267913A1 (en) 2007-12-06
JP2009538139A (ja) 2009-11-05
EP2021486B2 (fr) 2022-01-26
JP2016019537A (ja) 2016-02-04
KR20090018978A (ko) 2009-02-24
JP2018007688A (ja) 2018-01-18
CA2652801C (fr) 2018-05-22
EP2021486B1 (fr) 2014-10-29
US9765363B1 (en) 2017-09-19
CN105331518A (zh) 2016-02-17
MX2008014970A (es) 2009-02-18
JP2020018319A (ja) 2020-02-06
MX293430B (es) 2011-12-09
ZA200809957B (en) 2010-02-24
DK2021486T3 (en) 2014-12-15
JP6673879B2 (ja) 2020-03-25
BRPI0712508B1 (pt) 2018-04-10
MY163029A (en) 2017-07-31
EP2021486B9 (fr) 2022-04-20
WO2007139924A3 (fr) 2008-02-07
DK2021486T4 (da) 2022-03-07
ES2527876T3 (es) 2015-02-02

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